Suppression of B-cell apoptosis in transgenic animals expressing humanized immunoglobulin

ABSTRACT

The invention provides a novel approach to increase immunoglobulin expression in non-human transgenic animals. For instance, the invention provides a method to increase humanized immunoglobulin production in animals genetically engineered to express one or several human or humanized immunoglobulin transloci. This can be done by overexpressing the apoptosis inhibitor, i.e. a rabbit bcl-2, whose expression is driven by a B-cell specific promoter specifically in the B-cell of the animal, thereby enhancing the survival of B-cells. This invention further relates to a method for selectively enhancing the survival of exogenous B-cells, that is B-cells expressing any immunoglobulin transgene locus, over the survival of endogenous B-cells that do not express the transgene locus. Selectivity is achieved by expressing the apoptosis-inhibitor only within exogenous B-cells, that is, by coupling exogenous immunoglobulin expression with apoptosis inhibitor expression. This latter method allows for increased expression and production of the transgene encoded product(s) over the endogenously produced immunoglobulin of the transgenic animal. The invention also provides a novel apoptosis-inhibitor, rabbit bcl-2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application filed under 37 CFR 1.53(b),claiming priority under U.S.C. Section 19(e) to U.S. Provisional PatentApplication Ser. No. 60/705,305 filed Aug. 3, 2005.

FIELD OF THE INVENTION

This invention relates to methods for enhancing the survival of B-cellsin animals undergoing short-term lymphopoiesis. This invention furtherrelates to methods for enhancing the survival of B-cells of transgenicanimals expressing an exogenous immunoglobulin or immunoglobulin chaintransgene locus for increasing the production of immunoglobulins. Thisinvention further relates to a method for selectively enhancing thesurvival of exogenous B-cells expressing any immunoglobulin transgenelocus over endogenous B-cells that do not express the transgene locus byselectively expressing any apoptosis-inhibitor only within exogenousB-cells expressing the transgene-encoded immunoglobulin, but not withinB-cells expressing endogenous immunoglobulin. This method allows for theincreased expression and production of the transgene encoded product(s)over the endogenously produced immunoglobulin of the transgenic animal.The invention also provides a novel apoptosis-inhibitor, rabbit bcl-2.

BACKGROUND ART

The generation of mice expressing human-mouse chimeric antibodies hasbeen described by Pluschke et al., Journal of Immunological Methods 215:27-37 (1998). The generation of mice expressing human immunoglobulinpolypeptides has been described by Neuberger et al., Nature 338: 350-2(1989); Lonberg et al., Int. Rev. Immunol. 13(1):65-93 (1995); andBruggemann et al., Curr. Opin. Biotechnol., 8(4): 455-8 (1997).Generation of transgenic mice using a BAC clone has been described byYang et al., Nat. Biotechnol. 15: 859-65 (1997). The generation of cowsexpressing human antibodies has been described by Kuroiwa et al., NatureBiotech 20(9): 889-894 (2002).

Transgenesis in animals has been described by Wall R J, Theriogenology57(1): 189-201 (2002). The generation of transgenic rabbits has beendescribed by Fan, J. et al., Pathol Int. 49: 583-94 (1999); and Brem etal., Mol. Reprod. Dev. 44: 56-62 (1996). The production of transgenicchicken has been described by Etches et al., Methods in MolecularBiology 62: 433-450 (1997); and Pain et al., Cells Tissues Organs165(3-4): 212-9 (1999); and Sherman et al., Nature Biotech 16:1050-1053(1998).

Rabbits with impaired immunoglobulin expression have been described byChen et al., J. Immunol. 150:2783-2793 (1993); and Lamoyi E, and Mage RG., J. Exp. Med. 162:1149-1160 (1985). A gamma-globulinemic chicken hasbeen described by Frommel et al., J. Immunol. 105(1): 1-6 (1970); andBenedict et al., Adv. Exp. Med. Biol. 88(2): 197-205 (1977).

The cloning of animals from cells has been described by T. Wakayama etal., Nature 394:369-374 (1998); J. B. Cibelli et al., Science280:1256-1258 (1998); J. B. Cibelli et al., Nature Biotechnology16:642-646 (1998); A. E. Schnieke et al., Science 278: 2130-2133 (1997);and K. H. Campbell et al., Nature 380: 64-66 (1996). Nuclear transfercloning of rabbits has been described by Stice et al., Biology ofReproduction 39: 657-664 (1988); Challah-Jacques et al., Cloning andStem Cells 8(4):295-299 (2003).

The production of non-human transgenic animals expressing human(ized)immunoglobulin transloci and the production of antibodies from suchtransgenic animals have been described in detail in PCT Publication Nos.WO 92/03918, WO 02/12437, and in U.S. Pat. Nos. 5,545,807, 5,814,318;and 5,570,429. Homologous recombination for chimeric mammalian hosts isexemplified in U.S. Pat. No. 5,416,260. A method for introducing DNAinto an embryo is described in U.S. Pat. No. 5,567,607. Maintenance andexpansion of embryonic stem cells is described in U.S. Pat. No.5,453,357.

The cleavage activities of viral proteins containing 2A peptidesequences have been described by Palmenberg et al., Virology 190:754-762(1992); Ryan et al., J Gen Virol 72:2727-2732 (1991); Donnelly et al., JGen Virol 82:1027-1041 (2001); Donnelly et al., J Gen Virol 82:1013-1025(2001); Szymaczak et al., Nature Biotech 22(5):589-594 (2004).

So far, studies of the relative contribution of cell survival mechanismsregulated by the apoptosis inhibitor bcl-2, have been performed mainlyin mice. The effect of bcl-2 expression on cell survival has beendescribed by McDonnell et al., Cell, 57:79-88, (1989); Strasser et al.,Current Topics in Microbiology and Immunology, 166:175-181, (1990);Knott et al., Hybridoma, 15 (5):365-371, (1996); Smith et al., J. Exp.Med., 191(3):475-784 (2000); Strasser et al., PNAS, 88:8661-8665, (1991)and Kumar et al., Immunology Letters, 65:153-159, (1999). The effect ofthe apoptosis inhibitor bcl-x_(L) expression on cell survival has beendescribed by Takahashi et al., J. Exp. Med., 190(3): 399-409 (1999).

Mechanisms of B-cell development such as continuous and short-term Blymphopoiesis have been reviewed in Lanning D, Osborne B A, Knight, KL., Immunoglobulin genes and generation of antibody repertoires inhigher vertebrates: a key role of GALT. Molecular Biology of B-cells.Alt F. W., Honjo T, Nueberger, M. S., Eds. Elsevier London, p 443(2004); and Flajnik M. F., Comparative analysis of immunoglobulin genes:surprises and portents. Nat. Rev. Immunol. 2:688, (2002).

Since production of antibodies in larger transgenic animals likerabbits, chickens, sheep and cows is favored from the standpoint ofantibody yield, creation of larger founder animals with B-cell apoptosisinhibition expressing higher amounts of transgene-encoded products ishighly desirable. However, B-cell development differs significantly inspecies undergoing short-term lymphopoiesis (like rabbits, chickens,sheep and cows) relative to animals characterized by continuous Blymphopoiesis (like mice). Thus, it is unclear if apoptosis inhibitorscan be used with the same success in animals undergoing short-termlymphopoiesis as in the more extensively studied animals with continuousB lymphopoiesis, or, what the impact of apoptosis inhibitors on antibodyproduction and/or antibody affinities will be.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a polypeptide comprising a novelapoptosis-inhibitor polypeptide, namely, the rabbit bcl-2 polypeptide ofSEQ ID NO: 5. In a particular embodiment, the invention provides achimeric molecule comprising the rabbit bcl-2 polypeptide of SEQ ID NO:5 fused to a heterologous amino acid sequence. In a further embodiment,the heterologous amino acid sequence is an epitope sequence. In anotherembodiment, the heterologous amino acid sequence is an immunoglobulinsequence. In yet another embodiment, the immunoglobulin sequence is anFc region of an immunoglobulin. The present invention also provides anucleotide sequences encoding the rabbit bcl-2 polypeptide of SEQ ID NO:5. In one aspect, the invention provides a vector, expression cassetteor transgenic expression construct comprising the nucleic acid moleculethat encodes the rabbit bcl-2 polypeptide. In another aspect, theinvention provides an isolated host cell transformed with the nucleicacid sequences encoding the rabbit bcl-2 polypeptide of SEQ ID NO: 5. Ina further aspect, the invention provides an isolated host celltransformed with the vector, expression cassette or transgenicexpression construct comprising the nucleic acid molecule that encodesthe rabbit bcl-2 polypeptide.

In some aspects, any apoptosis inhibitor gene can be used, for example,an apoptosis inhibitor selected from the group consisting of bcl-2,caspase-9-DN mutants, baculovirus p35, caspase-9S, crmA, z-VAD-fmk,z-DEVD-fmk, B-D-fmk, z-YVAD-fmk, Bcl-x_(L), MCl-1, XIAP, TIAP, KIAP,NAIP, cIAP1, cIAP2, API1, API2, API3, API4, HIAP1, HIAP2, MIHA, MIHB,MIHC, ILP, ILP-2, TLAP, survivin, livin, apollon, BRUCE, MLIAP, SODD andFLIP and variants thereof. In some specific embodiments, the apoptosisinhibitor gene may be a mammalian bcl-2 gene. In some preferredembodiments, the mammalian bcl-2 gene is selected from the groupconsisting of human bcl-2, mouse bcl-2 and rabbit bcl-2 of SEQ ID NO: 6.In a preferred embodiment, the bcl-2 is the rabbit bcl-2 of SEQ ID NO:5.

In one aspect, the invention provides a transgenic expression constructcomprising a nucleic acid molecule that encodes an apoptosis inhibitordriven by a B-cell specific promoter/enhancer and thus, is specificallyexpressed in B-cells.

In another aspect, the invention provides a transgenic expressionconstruct comprising a transgene encoding a fusion-protein comprisingpolypeptide sequences in the following order: a) an immunoglobulin orimmunoglobulin chain; b) a self-cleaving peptide; c) an apoptosisinhibitor; and optionally, d) a protease cleavage site between a) andb).

The present invention further provides a method for enhancing theexpression of an immunoglobulin or immunoglobulin chain in a transgenicanimal undergoing short-term lymphopoiesis, comprising introducing intothe transgenic animal undergoing short-term lymphopoiesis at least onetransgene construct comprising an apoptosis-inhibitor transgene drivenby a B-cell specific promoter/enhancer whereby apoptosis of the B-cellscarrying said transgene construct is inhibited and production of theimmunoglobulin or immunoglobulin chain is enhanced.

In a further aspect, the present invention provides a method forenhancing the expression of an immunoglobulin or immunoglobulin chain inthe short-term lymphopoietic transgenic animal that further comprisesintroducing into the transgenic animal at least one more transgeneencoding for an exogenous immunoglobulin or immunoglobulin chaintransgene locus. In this method, the two transgenes can both be presenton the same or on different transgenic expression vectors. In the lattercase, the different transgenic expression vectors can be introduced intothe transgenic animal either at the same time or sequentially.

The present invention also provides a method for selectively enhancingthe expression of an exogenous immunoglobulin or immunoglobulin chainwithin an exogenous B-cell of a non-human transgenic animal, comprisingintroducing into the animal, a transgene construct encoding afusion-protein comprising polypeptide sequences in the following order:a) an immunoglobulin or immunoglobulin chain; b) a self-cleavingpeptide; c) an apoptosis inhibitor, and, optionally; d) a proteasecleavage site between a) and b), whereby survival of the exogenous Bcell and exogenous immunoglobulin production are enhanced.

In any aspect, the protease cleavage site used in any of the transgenicconstructs or methods described above, is selected from the groupconsisting of sites for aspartic proteases, cysteine proteases,metalloproteases, serine proteases and threonine proteases. In preferredembodiments, the furin cleavage site is used.

In all aspects of the invention, the B-cell specific promoter/enhancermay be selected from the group consisting of promoters/enhancers ofCD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, Blimp-1, CD79b, mb-1,tyrosine kinase blk, VpreB, immunoglobulin heavy chain, immunoglobulinkappa light chain, immunoglobulin lambda-light chain and immunoglobulinJ-chain, or modifications thereof. In specific embodiments, the B-cellspecific promoter/enhancer is the immunoglobulin kappa light chain genepromoter or a modification thereof.

In all aspects of the invention, the preferred exogenousimmunoglobulin(s)/immunoglobulin chain transgene locus is thehuman/humanized immunoglobulin heavy and/or light chain sequence.

In all aspects of the invention, the self-cleaving peptide of theinvention can be obtained from viral 2A/2B or 2A-like/2B sequences.Thus, the virus may be selected from the group consisting of thepicornaviridae virus family, the equine rhinitis A (ERAV) virus family,the picornavirus-like insect virus family and from the type C rotavirusfamily. The virus may also be selected from the group consisting of thefoot and mouth disease virus (FMDV), the equine rhinitis A (ERAV) virus,and the Thosea asigna virus (TaV).

In a further aspect of the invention, the invention relates to non-humantransgenic animals comprising the transgenic constructs described above.For instance, the apoptosis inhibitor transgenes are preferablyintroduced into animals undergoing short-term lymphopoiesis. Theseinclude, but are not limited to, rabbits, birds, chickens, sheep, goats,cows, swine, horses and donkeys. These short-term lymphopoietic animalsmay further comprise transgenes, for instance, encoding animmunoglobulin(s)/immunoglobulin chain transgene. On the other hand, thefusion-protein encoding transgenes can be introduced into any non-humananimal.

Thus, in most aspects of the invention, unless specified, non-humananimals are selected from the group consisting of rodents (e.g. mice,rats), rabbits, birds (e.g. chickens, turkeys, ducks, geese, etc.),cows, pigs, sheep, goats, horses, donkeys and other farm animals. Insome aspects of the invention, the non-human transgenic animal caneither substantially stops antibody diversification by generearrangement early in life or substantially stops antibodydiversification within the first month of its life. In a specificembodiment, the non-human transgenic animal is the rabbit.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid alignment of the rabbit polypeptide sequence(SEQ ID NO: 5) with other bcl-2 molecules derived from other species(SEQ ID NOS: 11-20).

FIG. 2: SEQ ID NO: 1; A synthetic human bcl-2 apoptosis inhibitionvector under the control of the kappa 1 B cell specific promoter.

FIG. 3: SEQ ID NO: 2; DNA fragment encoding rabbit IgG M2-self cleavingpeptide F2A-human bcl2 fusion protein FRT rpsL-neo FRT.

FIG. 4: SEQ ID NO: 3; DNA fragment encoding rpsL-neo flanked with FRTand FRT2 sites.

FIG. 5: SEQ ID NO: 4; DNA fragment encoding rabbit IgM-M2-self cleavingpeptide F2A-codon optimized human bcl2 fusion protein flanked with FRTand FRT2 sites.

FIG. 6: SEQ ID NO: 5; The rabbit bcl-2 polypeptide sequence.

FIG. 7: SEQ ID NO: 6; DNA fragment encoding rabbit IgM-M2-self cleavingpeptide F2A-codon optimized human bcl2 fusion protein.

FIG. 8: SEQ ID NO: 7; DNA fragment encoding rabbit IgM-M2-self cleavingpeptide F2A-human bcl2 fusion protein

FIG. 9: SEQ ID NO: 8; DNA fragment encoding an IgG-M2-self cleavingpeptide F2A-codon optimized human bcl2 fusion protein

FIG. 10: SEQ ID NO: 9; DNA fragment encoding rabbit IgM-M2-furincleavage site-self cleaving peptide F2A-human bcl2 fusion protein

FIG. 11: SEQ ID NO: 10; DNA fragment encoding rabbit IgG-M2-furincleavage site-self cleaving peptide F2A-codon optimized human bcl2fusion protein

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“B-cells” are defined as B-lineage cells that are capable of undergoingrearrangement of immunoglobulin gene segments and expressingimmunoglobulin genes at some stage in their life cycle. These cellsinclude, but are not limited to, early pro-B-cells, late pro-B-cells,large pre-B-cells, small pre-B-cells, immature B-cells, mature B-cells,memory B-cells, plasma cells, etc.

“Apoptosis-inhibitors” refer to a molecule or substance the presence orexpression of which provides a reduction of apoptosis in target cells,regardless of the underlying mechanism. Preferably, theapoptosis-inhibitor reduces apoptosis of a target cell by at least about50%, or at least about 60%, or at least about 70%, or at least about75%, or at least about 80%, or at least about 85%, or at least about90%, or at least about 95% relative to apoptosis in the absence of theinhibitor.

The term “human Ig gene translocus or locus or segment” as used hereinincludes both naturally occurring sequences of a human Ig gene locus ora segment thereof, degenerate forms of naturally occurring sequences ofa human Ig gene locus or segments thereof, as well as syntheticsequences that encode a polypeptide sequence substantially identical toa polypeptide encoded by a naturally occurring sequence of a human Iggene locus or a segment thereof. In this context, by “substantially” ismeant the degree of amino acid sequence identity is preferably at leastabout 85%-95%, or more preferably at least about 90%-95%, or even morepreferably at least about 95%, or most preferably at least about 98%. Ina particular embodiment, the human Ig gene segment renders theimmunoglobulin molecule non-immunogenic in humans. Here, the terms“human or humanized immunoglobulin (Ig) heavy and/or light chain locus”or “human or human(ized) Ig locus” are used interchangeably.

The terms “human antibody” and “human immunoglobulin” are used herein torefer to antibodies and immunoglobulin molecules comprising fully humansequences.

The terms “humanized antibody” and “humanized immunoglobulin,” as usedherein, mean an immunoglobulin molecule comprising at least a portion ofa human immunoglobulin polypeptide sequence (or a polypeptide sequenceencoded by a human immunoglobulin gene segment). The humanizedimmunoglobulin molecules of the present invention can be isolated from atransgenic non-human animal engineered to produce humanizedimmunoglobulin molecules. Such humanized immunoglobulin molecules areless immunogenic to primates, especially humans, relative tonon-humanized immunoglobulin molecules prepared from the animal orprepared from cells derived from the animal. Humanized immunoglobulinsor antibodies include immunoglobulins (Igs) and antibodies that arefurther diversified through gene conversion and somatic hypermutationsin gene converting animals. Such humanized Ig or antibodies are not“human” since they are not naturally made by humans (since humans do notdiversify their antibody repertoire through gene conversion) and yet,the humanized Ig or antibodies are not immunogenic to humans since theyhave human Ig sequences in their structure.

“Transgenes or transgene constructs” are DNA fragments with sequencesencoding naturally or synthetic proteins normally not found in theanimal or cells of the animal. The term “transgene construct” is usedherein to refer to a polynucleotide molecule, which contains astructural “gene of interest” and other sequences facilitating genetransfer. This invention refers to at least two transgene constructs: 1)the rabbit bcl-2 apoptosis inhibitor transgene driven by a B-cellspecific promoter, and, 2) the human Ig locus-self-cleavingpeptide-apoptosis-inhibitor transgene construct.

“A transgenic expression vector or expression construct” refers to DNAfragments which encode, besides one or several transgene constructs ofthe invention, other regulatory DNA sequences required either fortemporal, cell specific, or enhanced expression of the transgene(s) ofinterest, within specific cells of the non-human transgenic animal.

The “human(ized) Ig locus—self-cleaving peptide—apoptosis-inhibitortransgene or transgene construct” refers to a transgene construct thatis transcribed into a single mRNA, which is translated into twopolypeptides, namely, the human(ized) immunoglobulin chain and anapoptosis-inhibitor, due to a self-cleaving mechanism discussed below.

The term “self-cleaving peptide” as used herein refers to a peptidesequence that is associated with a cleavage activity that occurs betweentwo amino acid residues within the peptide sequence itself. For example,in the 2A/2B peptide or in the 2A/2B-like peptides, cleavage occursbetween the glycine residue on the 2A peptide and a proline residue onthe 2B peptide. This occurs through a ‘ribosomal skip mechanism’ duringtranslation wherein, normal peptide bond formation between the 2Aglycine residue and the 2B proline residue of the 2A/2B peptide isimpaired, without affecting the translation of the rest of the 2Bpeptide. Such ribosomal skip mechanisms are well known in the art andare known to be used by several viruses for the expression of severalproteins encoded by a single messenger RNA.

The terms “endogenous Ig (immunoglobulin)-expressing B-cells” and“endogenous B-cells” are used interchangeably, and refer to thoseB-cells that express the animal's endogenous immunoglobulin locus.

The terms “exogenous Ig (immunoglobulin)-expressing B-cells” and“exogenous B cells” refer to those B-cells of a non-human animal thatundergo productive rearrangement of an exogenous human(ized) Igtranslocus introduced into such B-cells. The human(ized) Ig locus isintroduced into such B-cells as a separate expression construct or aspart of the same expression construct also encoding theapoptosis-inhibitor. Productive rearrangement of the human(ized) Iglocus results in the expression of the human(ized) Ig. and the transgeneencoded apoptosis-inhibitor. As a result, apoptosis in B-cellsexpressing exogenous immunoglobulin is inhibited and cell survival isenhanced.

By “B-cell specific expression of the apoptosis inhibitor gene” ismeant, expression of the apoptosis inhibitor gene product preferablywithin immune cells, more preferably within B-cells. Specific expressionof the apoptosis inhibitor gene within immune cells or B-cells isachieved using immune-specific or preferably, using B-cell specificpromoters to drive the expression of the apoptosis inhibitor gene.

By “selective expression of the apoptosis-inhibitor” is meant,expression of the apoptosis-inhibitor gene product preferentially withinexogenous B-cells rather than within endogenous B cells expressing thenative immunoglobulins of the transgenic animal. Preferably, theexpression level of the apoptosis-inhibitor is at least about 2-fold,more preferably at least about 5-fold, even more preferably at leastabout 10-fold, most preferably at least about 50-fold more in exogenousB-cells as compared to expression in endogenous B-cells.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. The term “antibody” is used herein in the broadest senseand specifically covers, without limitation, monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments so long as they exhibit the desired specificity.

The term “Ig gene segment” as used herein refers to segments of DNAencoding various portions of an Ig molecule, which are present in thegermline of animals and humans, and which are brought together inB-cells to form rearranged Ig genes. Thus, Ig gene segments as usedherein include V gene segments, D gene segments, J gene segments and Cregion gene segments. Functional rearrangement of VDJ or VJ segmentsresults in the expression of immunoglobulin heavy or light chain.

The terms “antibody diversity” and “antibody repertoire” are usedinterchangeably, and refer to the total of all antibody specificitiesthat an organism is capable of expressing.

An Ig locus having the capacity to undergo gene rearrangement and geneconversion is also referred to herein as a “functional” Ig locus, andthe antibodies with a diversity generated by a functional Ig locus arealso referred to herein as “functional” antibodies or a “functional”repertoire of antibodies.

The term “monoclonal antibody” is used to refer to an antibody moleculesynthesized by a single clone of B-cells.

The term “polyclonal antibody” is used to refer to a population ofantibody molecules synthesized by a population of B-cells.

The terms “polynucleotide” and “nucleic acid” are used interchangeably,and, when used in singular or plural, generally refer to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas defined herein include, without limitation, single- anddouble-stranded DNA, DNA including single- and double-stranded regions,single- and double-stranded RNA, and RNA including single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or includesingle- and double-stranded regions. In addition, the term“polynucleotide” as used herein refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The strands in such regionsmay be from the same molecule or from different molecules. The regionsmay include all of one or more of the molecules, but more typicallyinvolve only a region of some of the molecules. One of the molecules ofa triple-helical region often is an oligonucleotide. The term“polynucleotide” specifically includes cDNAs. The term includes DNAs(including cDNAs) and RNAs that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritiated bases, are included within the term“polynucleotides” as defined herein. In general, the term“polynucleotide” embraces all chemically, enzymatically and/ormetabolically modified forms of unmodified polynucleotides, as well asthe chemical forms of DNA and RNA characteristic of viruses and cells,including simple and complex cells.

The term “non-human (transgenic) animal” as used herein includes, but isnot limited to, mammals such as, for example, non-human primates,rodents (e.g. mice and rats), non-rodent mammals, such as, for example,rabbits, pigs, sheep, goats, cows, pigs, horses and donkeys, and birds(e.g., chickens, turkeys, ducks, geese and the like). The term“non-primate animal” as used herein includes, but is not limited to,mammals other than primates, including but not limited to the mammalsspecifically listed above.

The phrase “animals which create antibody diversity substantially bygene conversion and/or somatic hypermutation to create primary antibodyrepertoires” or “gene converting animals” and their grammaticalequivalents, are used to refer to such animals in which the predominantmechanism of antibody diversification is gene conversion and/orhypermutation as opposed to gene rearrangement. Such animals include,but are not limited to, rabbits, birds (e.g., chickens, turkeys, ducks,geese and the like), cows and pigs. Particularly preferred non-humananimals are rabbits and chickens.

By animals “stopping antibody gene rearrangement early in life” is meantthose animals where the rearrangement of immunoglobulin genes stopstypically within the first month of life. Examples of such animals are,without limitation, rabbits, birds (e.g. chickens), sheep, goats,cattle, swine and horses.

DETAILED DESCRIPTION

This invention, at least in part, is based on the recognition that theproduction of immunoglobulin (including immunoglobulin chains) in anon-human transgenic animal undergoing short-term lymphopoiesis can besignificantly increased by expressing an apoptosis inhibitor in the Bcells of the animal. As a result, the survival of B cells is enhancedand the production of immunoglobulin is increased.

The invention is further based on the identification on a novelapoptosis inhibitor, rabbit bcl-2. Accordingly, in one embodiment, theinvention relates to methods for increasing immunoglobulin expression innon-human transgenic animals by overexpressing rabbit bcl-2 in theanimals' B cells, using a B-cell specific promoter, thereby enhancingB-cell survival.

This invention further relates to a method for selectively enhancing thesurvival of exogenous B-cells, that is, B-cells expressing animmunoglobulin transgene locus, over the survival of endogenous B-cellsthat do not express such a transgene locus in non-human animals,undergoing short-term lymphopoiesis. Selectivity is achieved by couplingexogenous immunoglobulin expression with apoptosis inhibitor expression.In endogenous B-cells, the apoptosis inhibitor is not expressed andhence, apoptosis is not inhibited. Such selective expression results inthe preferential production of the transgene expressed immunoglobulinover the endogenously produced immunoglobulin of the transgenic animal.

Overexpression of bcl-2 apoptosis inhibitors (other than the rabbitsequence first disclosed herein) has mainly been studied in mice whichshowed amplified and prolonged antibody responses to immunization due toa great excess of B lymphocytes, immunoglobulin-secreting cells, andserum immunoglobulins, attributable to increased longevity of B-lineagecells and antigen-specific memory B cells; McDonnell et al., Cell,57:79-88, (1989); Strasser et al., Current Topics in Microbiology andImmunology, 166:175-181, (1990); Knott et al., Hybridoma, 15(5):365-371, (1996); Smith et al., J. Exp. Med., 191(3):475-784 (2000);Strasser et al., PNAS, 88:8661-8665, (1991) and Kumar et al., ImmunologyLetters, 65:153-159, (1999).

Apoptosis of targeted B-cell populations occurs routinely throughoutB-cell development. Two major strategies for B-cell development havebeen identified through the study of different species: continuous Blymphopoiesis, as found in mice and humans, and short-term Blymphopoiesis followed by expansion in gut-associated lymphoid tissue(GALT), as found in chickens, rabbits, sheep and cows (reviewed inLanning D, Osborne B A, Knight, K L., Immunoglobulin genes andgeneration of antibody repertoires in higher vertebrates: a key role ofGALT. Molecular Biology of B-cells. Alt F. W., Honjo T, Nueberger, M.S., Eds. Elsevier London, p 443 (2004); and Flajnik M. F., Comparativeanalysis of immunoglobulin genes: surprises and portents. Nat. Rev.Immunol. 2:688, (2002)).

In species where continued B lymphopoiesis occurs, B-cells developprimarily in the bone marrow and fetal liver, and immunoglobulin genesdiversify on-site through the process of combinatorial V(D)J joining.Most of the peripheral blood lymphocytes in such species are IgM⁺, IgD⁺,or naïve B-cells with undiversified VDJ and VJ genes, even in adults.Thus, there may be lesser pressure to produce a B-cell compartmentquickly in animals with continued B lymphopoiesis since new B-cells withnovel antigenic specificities are produced continuously.

In contrast, in the GALT (gut-associated lymphoid tissue) species whereB lymphopoiesis is brief, an initial pool of B-cells is formed early inlife in tissues such as the yolk sac and spleen, and thereafter,immunoglobulin (Ig) genes diversify in the GALT. Because B lymphopoiesisarrest is rapid, this initial B-cell compartment must expand anddiversify quickly to generate antibodies with biologically relevantspecificities. For instance, somatic diversification of Ig genes beginseven before birth in chickens, sheep and cows. As a consequence, nearlyall of the VDJ genes within the peripheral blood lymphocytes of adultrabbits for example, are highly diversified and lack naïve B-cells. Yet,the adult rabbit is very capable of mounting primary antibody responsesto previously unseen antigens. That is because B-cells migrating fromthe GALT to the periphery appear to be of the primary B-cell repertoire,and even though their VDJ genes are already diversified, theselong-lived and/or self-renewing B-cells can maintain the functionalantibody repertoire. It is also likely that, as in the case of rabbits,exogenous antigenic stimulation helps drive the diversification of theantibody repertoire in species with short B lymphopoiesis.

In normal mice, during primary T cell-dependent immune responses,somatic mutations of Ig V region genes occurs in germinal-center-B-cellsthus generating variant B-cells that express immunoglobulins withaltered affinities for the antigen. Variants with improved affinity arepositively selected through the inhibition of apoptosis and eventually,such high affinity B-cells make up the majority of the antigen specificmemory and antibody-forming B-cell populations. B-cells with a lowaffinity receptor fail to receive such antigen-dependent survivalsignals and undergo apoptosis. Such an increase in high-affinityB-cells, within memory and antibody-forming B-cell populations, isreferred to as affinity maturation.

In bcl-2 transgenic mice, overexpression of bcl-2 results in theprevention of apoptosis not only of high affinity B-cells, but also oflow affinity B-cells. Bcl-2 overexpressing mice have an excessive numberof memory B-cells that are not affinity selected. In contrast, thestringent selection of high-affinity bone marrow antibody-forming cellsin the bcl-2 mouse is not influenced by the bcl-2 transgene and theirnumbers remain unchanged compared to controls.

While the effects of overexpression of bcl-2 on B-cell survival anddevelopment in other animals undergoing continuous B lymphopoiesis maybe similar to that in mice, its role in the development of memory and/orantibody-forming B-cells of animals undergoing short-term Blymphopoiesis is unclear.

Therefore, the present invention is directed to methods foroverexpressing apoptosis inhibitors, particularly in animals withshort-term B lymphopoiesis like rabbits, birds, chickens, sheep, goats,cows, swine, horses and donkeys and enhancing B-cell survival in suchtransgenic animals. In addition, when these animals further express anIg translocus, expression of the Ig translocus is enhanced or prolongedand since these are larger animals, their antibody yields should also begreater. Thus, this invention aims at creating larger founder animalsproducing higher amounts exogenous immunoglobulins through enhancedB-cell survival.

In one aspect, the present invention is directed to transgenicconstructs useful for enhancing the survival of B-cells. Transgenes ortransgene constructs are DNA fragments with sequences encoding for one,or several, natural or synthetic proteins not normally found in theanimal or cells of the animal. The DNA fragment(s) may be introducedinto the animal's genome by a variety of techniques includingmicroinjection of pronuclei, transfection, nuclear transfer cloning,sperm-mediated gene transfer, testis-mediated gene transfer, and thelike.

In one embodiment, the transgene construct comprises the nucleic acidmolecule encoding the apoptosis inhibitor, rabbit bcl-2 polypeptide. By“nucleic acid molecule encoding the apoptosis inhibitor” is meant thenative DNA sequence, as well as any codon optimized DNA sequence whichencodes for the a polypeptide sequence identical to the native DNAsequence, but which has a different DNA sequence based on codondegeneracy. This concept is discussed in detail below. In anotherembodiment, the transgene construct comprises the nucleic acid moleculeencoding any apoptosis inhibitor. The apoptosis-inhibitor gene, such asthe rabbit bcl-2 gene or the human bcl-2 gene, is preferably expressedin B-cells of the transgenic animal by means of an immune-specificpromoter, preferably a B-cell specific promoter. Therefore,apoptosis-inhibitor expression is enhanced preferably within B-cellsalone leading to enhanced B-cell survival in the non-human transgenicanimal. By “B-cell specific promoter” is meant the promoter/enhancerssequence of any B-cell specific genes, and/or variants or engineeredportions thereof, that normally controls the expression of genesexpressed in a B-cell, examples of which include, but are not limitedto, promoters/enhancers of CD19, CD20, CD21, CD22, CD23, CD24, CD40,CD72, Blimp-1, CD79b (also known as B29 or Ig beta), mb-1 (also known asIg alpha), tyrosine kinase blk, VpreB, immunoglobulin heavy chain,immunoglobulin kappa light chain, immunoglobulin lambda-light chain,immunoglobulin J-chain, etc. In a preferred embodiment, the kappa lightchain promoter/enhancer drives the B-cell specific expression of therabbit bcl-2 apoptosis-inhibitor gene.

In yet another embodiment, the transgene construct comprising thenucleic acid molecule encoding the apoptosis inhibitor is coexpressedwith a transgene construct comprising an exogenous immunoglobulin orimmunoglobulin (Ig) chain transgene locus. In this embodiment, both theIg transgene locus and the apoptosis inhibitor transgene may be presenton the same transgenic expression vector or on two different transgenicexpression vectors. In the latter case, the two transgenic expressionvectors may be introduced into the non-human transgenic animal either atthe same time or sequentially.

The present invention also provides transgene constructs comprising achimeric transgene that encodes for a fusion protein comprising atransgene encoding a fusion-protein comprising polypeptide sequences inthe following order: a) an immunoglobulin or immunoglobulin chain; b) aself-cleaving peptide; c) an apoptosis inhibitor; and optionally, d) aprotease cleavage site between a) and b). Here, the expression of theapoptosis inhibitor is linked or coupled to the expression of theimmunoglobulin heavy or light chain using mechanisms discussed below.This transgenic construct is also referred to as the Ig locus-proteasecleavage site-self cleaving peptide-apoptosis inhibitor construct. Inthis construct, a protease cleavage site is optionally added tofacilitate the removal of the F2A self-cleaving peptide sequence fromthe immunoglobulin; for instance, from the M2-exon of the Ig, to preventany potential interference of the F2A peptide sequence with signaling(and therefore B-cell development). The protease cleavage sites can berecognized by any constitutively expressed proteases. Protease cleavagesites useful herein include, but are not limited to, aspartic proteases,cysteine proteases, metalloproteases, serine proteases threonineproteases, etc. In a preferred embodiment, the protease cleavage site isthe furin cleavage site.

The chimeric transgenes described above comprises DNA sequences encodingfor a self cleaving peptide (for example, 2A peptide or 2A-likepeptide). Insertion of a self-cleaving peptide-encoding sequence betweenthe immunoglobulin-encoding sequence and an apoptosis-inhibitor sequencein the transgene results in production of one messenger RNA. Translationof this mRNA, however, results in two separate proteins, theimmunoglobulin(s) and the apoptosis-inhibitor, due to the peptide'sself-cleaving mechanism. Therefore, expression of theapoptosis-inhibitor can be coupled to the functional rearrangement ofVDJ or VJ segments.

In one such embodiment of the invention, the self-cleaving is mediatedby 2A/2B peptides, or 2A-like/2B sequences of viruses that include thepicornaviridae virus family, the equine rhinitis A (ERAV) virus family,the picornavirus-like insect virus family or from the type C rotavirusfamily. The picornaviridae virus family includes the entero-, rhino-,cardio- and aphtho- and foot-and-mouth disease (FMDV) viruses. Thepicomavirus-like insect virus family includes viruses such as theinfectious flacherie virus (IFV), the Drosophila C virus (DCV), theacute bee paralysis virus (ABPV) and the cricket paralysis virus (CrPV)and the insect virus Thosea asigna virus (TaV). The type C rotavirusfamily includes the bovine, porcine and human type C rotaviruses. Infurther embodiments, the cleavage sequences may include 2A-like/2Bsequences from either the poliovirus, rhinovirus, coxsackie virus,encephalomyocarditis virus (EMCV), mengovirus, the porcineteschovirus-1, or the Theiler's murine encephalitis virus (TMEV), etc.In a preferred embodiment, the self-cleaving protein sequence is eitherthe 2A/2B peptide of the foot and mouth disease virus (FMDV), the equinerhinitis A (ERAV) virus, or the Thosea asigna virus (TaV); Palmenberg etal., Virology 190:754-762 (1992); Ryan et al., J Gen Virol 72:2727-2732(1991); Donnelly et al., J Gen Virol 82:1027-1041 (2001); Donnelly etal., J Gen Virol 82:1013-1025 (2001); Szymaczak et al., Nature Biotech22(5):589-594 (2004). Thus, using the self-cleaving peptide, expressionof the apoptosis inhibitor gene is linked or coupled to the expressionof the Ig translocus within exogenous B-cells. Selective survival ofexogenous B-cells over endogenous B-cells results in reduced endogenousimmunoglobulin production but in a corresponding increase in productionof the Ig translocus encoded polypeptide/protein.

While bcl-2 is discussed as a prototype of apoptosis-inhibitors, otherapoptosis-inhibitors are also included for use in the chimeric transgeneconstruct. These include, without limitation, caspase-9 dominantnegative (caspase-9-DN) mutants, baculovirus p35, caspase-9S, crmA,z-VAD-fmk, z-DEVD-fmk, B-D-fmk, and z-YVAD-fmk, other bcl-2 familymembers like Bcl-x_(L), Mcl-1, etc., inhibitors of proapoptoticmolecules like Bax, Bak, Bad, inhibitors of “BH3 domain only” moleculeslike Bid, Bim, PUMA, Noxa, etc., other endogenous caspase inhibitorslike IAP (inhibitor of apoptosis proteins) including, but not limited toXIAP, TIAP, KIAP, NAIP, cIAP1, cIAP2, API1, API2, API3, API4, HIAP1,HIAP2, MIHA, MIHB, MIHC, ILP, ILP-2, TLAP, survivin, livin, apollon,BRUCE, and MLIAP, etc., proteins like SODD and FLIP, etc. involved inthe down-regulation of death receptors and variants thereof. In aspecific embodiment, the apoptosis inhibitor gene may be a mammalianbcl-2 gene and in preferred embodiments, the mammalian bcl-2 gene isselected from the group consisting of human bcl-2, mouse bcl-2 andrabbit bcl-2 of SEQ ID NO: 5. In a preferred embodiment, the rabbitbcl-2 gene of SEQ ID NO: 5 is used.

In yet another aspect of the invention, the transgene encodesimmunoglobulin heavy chains and/or immunoglobulin light chains or partsthereof. The loci can be in germline configuration or in a rearrangedform. The coding sequences or parts thereof may code for humanimmunoglobulins resulting in the expression of human(ized) antibodies.

The transgene(s) encoding human(ized) antibodies contain(s) an Ig locusor a large portion of an Ig locus, containing one or several human Igsegments (e.g., a human Ig V, D, J or C gene segment). Alternatively,the transgene is a human immunoglobulin locus or a large portionthereof. The transgene containing such a human Ig locus or such modifiedIg locus or modified portion of an Ig locus, also referred to herein as“a human(ized) Ig translocus”, is capable of undergoing generearrangement in the transgenic non-human animal thereby producing adiversified repertoire of antibodies having at least a portion of ahuman immunoglobulin polypeptide sequence.

Immunoglobulin heavy and light chain genes comprise several segmentsencoded by individual genes and separated by intron sequences. Thusgenes for the human immunoglobulin heavy chain are found on chromosome14. The variable region of the heavy chain (VH) comprises three genesegments: V, D and J segments, followed by multiple genes coding for theC region. The V region is separated from the C region by a large spacer,and the individual genes encoding the V, D and J segments are alsoseparated by spacers.

There are two types of immunoglobulin light chains: κ and λ. Genes forthe human κ light chain are found on chromosome 2 and genes for thehuman λ light chain are found on chromosome 22. The variable region ofantibody light chains includes a V segment and a J segment, encoded byseparate gene segments. In the germline configuration of the κ lightchain gene, there are approximately 100-200 V region genes in lineararrangement, each gene having its own leader sequence, followed byapproximately 5 J gene segments, and C region gene segment. All Vregions are separated by introns, and there are introns separating theV, J and C region gene segments as well.

Additionally, the vectors containing either of the transgene constructsdescribed above may further contain DNA sequences coding for antibioticselection markers like gentamycin, neomycin or kanamycin etc. and/orother conventional components of expression vectors.

The present invention provides methods for enhancing the expression ofimmunoglobulins in a non-human transgenic animal undergoing short-termlymphopoiesis comprising introducing into the transgenic animalundergoing short-term lymphopoiesis, at least one transgene constructcomprising an apoptosis-inhibitor transgene driven by a B-cell specificpromoter/enhancer. Thus, apoptosis of such B-cells with the transgeneconstruct is inhibited and production of the immunoglobulin orimmunoglobulin chain is enhanced. In a further embodiment of thismethod, the non-human transgenic animal undergoing short-termlymphopoiesis may further comprise an exogenous immunoglobulin(s) orimmunoglobulin chain transgene locus. This results in higher yields ofthe exogenous immunoglobulin which can greatly simplify antibodypurification and production. In this instance, the apoptosis-inhibitorgene may be introduced, either, as part of a transgenic expressionconstruct that also introduces the Ig translocus, or on differenttransgenic constructs.

The invention further provides another method for selectively enhancingthe expression of an exogenous immunoglobulin(s)/immunoglobulin chainwithin an exogenous B-cell of a non-human transgenic animal, whereexpression of the exogenous immunoglobulin(s)/immunoglobulin chain andan apoptosis inhibitor transgene within the exogenous B-cell is coupled.Correspondingly, there is no expression of the apoptosis inhibitor inendogenous B-cells, or B-cells not expressing the Ig translocus. Due toproductive rearrangement of the exogenous immunoglobulin translocus andan increase in exogenous B-cell survival, transgene-encodedimmunoglobulin production is increased over endogenous immunoglobulinproduction. Thus, survival of the exogenous B cell is enhanced andexogenous immunoglobulin(s)/immunoglobulin chain production is alsoenhanced.

The present invention further provides nucleic acid sequences thatencode for proteins, polypeptides or peptide sequences for rabbit bcl-2,which is an apoptosis-inhibitor. It is also contemplated that a givennucleic acid sequence for rabbit bcl-2 may be represented by naturalvariants that have slightly different nucleic acid sequences but,nonetheless, encode the same protein. Furthermore, the term functionallyequivalent codon is used herein to refer to codons that encode the sameamino acid, for example, as the six codons for arginine or serine, andalso refers to codons that encode biologically equivalent amino acids,as discussed herein.

The rabbit bcl-2 DNA segments used in the present invention encompassbiologically functional equivalent modified polypeptides and peptides.Such sequences may arise as a consequence of codon redundancy andfunctional equivalency that are known to occur naturally within nucleicacid sequences and the proteins thus encoded. Alternatively,functionally equivalent proteins or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by humanmay be introduced through the application of site-directed mutagenesistechniques, e.g., to introduce improvements to the antigenicity of theprotein, to reduce toxicity effects of the protein in vivo to a subjectgiven the protein, or to increase the efficacy of any treatmentinvolving the protein.

Allowing for the degeneracy of the genetic code, the inventionencompasses sequences that have at least about 50%, usually at leastabout 60%, more usually about 70%, most usually about 80%, preferably atleast about 90% and most preferably about 95% sequence identity to thenucleotide sequence of the rabbit bcl-2 gene or the human bcl-2 gene,respectively. These are also referred to as codon optimized sequencesand is discussed below under functionally equivalent codons.

The term biologically functional equivalent is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%; or more preferably, between about81% and about 90%; or even more preferably, between about 91% and about99% identical at the amino acid level are considered functionallyequivalent to the rabbit bcl-2 polypeptide, provided the biologicalactivity of the protein is maintained.

The term functionally equivalent codon is used herein to refer to codonsthat encode the same amino acid, such as the six codons for arginine orserine, and also refers to codons that encode biologically equivalentamino acids.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow.

In making such changes, the hydropathic index of amino acids may also beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1);glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2) glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+-0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within .+-.2is preferred, those that are within .+-.1 are particularly preferred,and those within .+-..5 are even more particularly preferred.

As outlined herein, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure (Johnson 1993). The underlying rationale behind the use ofpeptide mimetics is that the peptide backbone of proteins exists chieflyto orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples outlined above, to engineer second generation moleculeshaving many of the natural properties of apoptosis-inhibitors withaltered and improved characteristics.

Thus, variant nucleic acid sequences that encode for rabbit bcl-2 andfunctionally equivalent polypeptides of rabbit bcl-2 are useful asapoptosis-inhibitors in this invention.

The immune system's capacity to protect against infection rests in agenetic machinery specialized to create a diverse repertoire ofantibodies. Antibody-coding genes in B-cells are assembled in a mannerthat allows to countless combinations of binding sites in the variable(V) region. It is estimated that more than 10¹² possible bindingstructures arise from such mechanisms. In all animals, including humans,the antibody-making process begins by recombining variable (V),diversity (D) and joining (J) segments of the immunoglobulin (Ig) locus.Following this step, depending on the animal species, two generalmechanisms are used to produce the diverse binding structures ofantibodies.

In some animals, such as human and mouse, there are multiple copies ofV, D and J gene segments on the immunoglobulin heavy chain locus, andmultiple copies of V and J gene segments on the immunoglobulin lightchain locus. Antibody diversity in these animals is generated primarilyby gene rearrangement, i.e., different combinations of gene segments toform rearranged heavy chain variable region and light chain variableregion. In other animals (e.g., rabbit, birds, e.g., chicken, goose, andduck, sheep, goat, and cow), however, gene rearrangement plays a smallerrole in the generation of antibody diversity. For example, in rabbit,only a very limited number of the V gene segments, most often the V genesegments at the 3′ end of the V-region, is used in gene rearrangement toform a contiguous VDJ segment. In chicken, only one V gene segment (theone adjacent to the D region, or “the 3′ proximal V gene segment”), oneD segment and one J segment are used in the heavy chain rearrangement;and only one V gene segment (the 3′ proximal V segment) and one Jsegment are used in the light chain rearrangement. Thus, in theseanimals, there is little diversity among initially rearranged variableregion sequences resulting from junctional diversification. Furtherdiversification of the rearranged Ig genes is achieved by geneconversion a process in which short sequences derived from the upstreamV gene segments replace short sequences within the V gene segment in therearranged Ig gene. Additional diversification of antibody sequences maybe generated by hypermutation.

Immunoglobulins (antibodies) belong into five classes (IgG, IgM, IgA,IgE, and IgD, each with different biological roles in immune defense.The most abundant in the blood and potent in response to infection isthe IgG class. Within the human IgG class, there are four sub-classes(IgG1, IgG2, IgG3 and IgG4 isotypes) determined by the structure of theheavy chain constant regions that comprise the Fc domain. The F(ab)domains of antibodies bind to specific sequences (epitopes) on antigens,while the Fc domain of antibodies recruits and activates othercomponents of the immune system in order to eliminate the antigens.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by covalent disulfide bond(s), while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains (Chothia et al., J. Mol.Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, connected by three CDRs. TheCDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The creation of human-animal translocus allows for the creation oftransgenic animals that express diversified, high-affinity human(ized)(polyclonal) antibodies in high yields. In general, the humanization ofan immunoglobulin (Ig) locus in a non-human animal involves theintegration of one or more human Ig gene segments into the animal'sgenome to create human(ized) immunoglobulin loci. Thus, creation of ahuman(ized) Ig heavy chain locus involves the integration of one or moreV and/or D and/or J segments, and/or C region segments into the animal'sgenome. Similarly, the creation of a humanized Ig light chain locusinvolves the integration of one or more V and/or J segments, and/or Cregion segments into the animal's genome.

Regardless of the chromosomal location, the human(ized) Ig locus of thepresent invention has the capacity to undergo gene rearrangement andgene conversion and hypermutation in the non-human animal, therebyproducing a diversified repertoire of human(ized) Ig molecules. An Iglocus having the capacity to undergo gene rearrangement and geneconversion is also referred to as a “functional” Ig locus and theantibodies with a diversity generated by a functional Ig locus are alsoreferred to as “functional” antibodies or a “functional” repertoire ofantibody molecules.

In one aspect, animals in which diversification of the antibodyrepertoire stops early in life are useful in the current invention.B-cells develop from hematopoietic stem cells. Prior to antigenexposure, B-cells undergo a series of maturation steps the end productof which is a mature B-cell, which expresses a uniquemembrane-associated IgM and often IgD on its cell surface along withother cell surface signaling molecules. While in humans, antibodydiversification by gene rearrangement occurs throughout life, in otheranimals the diversification of antibody repertoire stops early in life,typically within the first month of life.

In one aspect of this invention, the animals to whom the DNA constructsof the invention can be administered include, but are not limited to,mammals (e.g. humans, non-human primates, rodents (e.g. mice and rats),non-rodents (e.g. rabbits, pigs, sheep, goats, cows, pigs, horses anddonkeys), and birds (e.g., chickens, turkeys, ducks, geese and thelike). The animals to whom the DNA constructs of the invention can beadministered include ‘gene converting animals’, that is, animals thatcreate antibody diversity substantially by gene conversion and/orsomatic hypermutation (for e.g. rabbits, birds, cows, swine, etc.), andanimals where antibody rearrangement stops early in life, that is,typically, within the first month of life (for e.g. rabbits, birds,sheep, goats, cattle, swine, horses, etc.).

Further, animals to whom the DNA constructs of the invention can beadministered also include any of the non-human animal described above,further carrying a transgene encoding an exogenous immunoglobulintranslocus, preferably, a human or humanized immunoglobulin heavy chainand/or immunoglobulin light chain sequence or parts thereof. Thetransgene locus can be either in the germline configuration or in arearranged form. Since the transgenes encode for human or humanizedimmunoglobulins or parts thereof, it results in the generation ofhumanized antibodies. Thus, for example, using the methods describedabove, enhanced production of humanized antibodies, can be generated intarget non-human animals using the rabbit bcl-2 apoptosis-inhibitordescribed in this invention.

According to the present invention, a transgenic animal capable ofmaking human(ized) immunoglobulins is made by introducing into arecipient cell or cells of an animal, one or more of the transgenicvectors described herein above, one of which carries a human(ized) Iglocus, and deriving an animal from the genetically modified recipientcell or cells.

The recipient cells may, for example, be from non-human animals whichgenerate antibody diversity by gene conversion and/or hypermutation,e.g., bird (such as chicken), rabbit, cows and the like. In suchanimals, the 3′proximal V gene segment is preferentially used for theproduction of immunoglobulins. Integration of a human V gene segmentinto the Ig locus on the transgene vector, either by replacing the 3′proximal V gene segment of the animal or by being placed in closeproximity of the 3′ proximal V gene segment, results in expression ofhuman V region polypeptide sequences in the majority of immunoglobulins.Alternatively, a rearranged human V(D)J segment may be inserted into theJ locus of the immunoglobulin locus on the transgene vector.

The transgenic vectors containing the genes of interest, namely, thehuman(ized) Ig locus and the apoptosis-inhibitor gene may be introducedinto the recipient cell or cells and then integrated into the genome ofthe recipient cell or cells by random integration or by targetedintegration.

For random integration, a transgenic vector containing a human(ized) Iglocus can be introduced into an animal recipient cell by standardtransgenic technology. For example, a transgenic vector can be directlyinjected into the pronucleus of a fertilized oocyte. A transgenic vectorcan also be introduced by co-incubation of sperm with the transgenicvector before fertilization of the oocyte. Transgenic animals can bedeveloped from fertilized oocytes. Another way to introduce a transgenicvector is by transfecting embryonic stem cells and subsequentlyinjecting the genetically modified embryonic stem cells into developingembryos. Alternatively, a transgenic vector (naked or in combinationwith facilitating reagents) can be directly injected into a developingembryo. Ultimately, chimeric transgenic animals are produced from theembryos which contain the human(ized) Ig transgene integrated in thegenome of at least some somatic cells of the transgenic animal.

In a particular embodiment, a transgene containing a human(ized) Iglocus is randomly integrated into the genome of recipient cells (such asfertilized oocyte or developing embryos) derived from animal strainswith an impaired expression of endogenous immunoglobulin genes. The useof such animal strains permits preferential expression of immunoglobulinmolecules from the human(ized) transgenic Ig locus. Examples for suchanimals include the Alicia and Basilea rabbit strains, as well asagammaglobinemic chicken strain, as well as immunoglobulin knock-outmice. Alternatively, transgenic animals with human(ized) immunoglobulintransgenes or loci can be mated with animal strains with impairedexpression of endogenous immunoglobulins. Offspring homozygous for animpaired endogenous Ig locus and a human(ized) transgenic Ig locus canbe obtained.

For targeted integration, a transgenic vector can be introduced intoappropriate animal recipient cells such as embryonic stem cells oralready differentiated somatic cells. Afterwards, cells in which thetransgene has integrated into the animal genome and has replaced thecorresponding endogenous Ig locus by homologous recombination can beselected by standard methods See for example, Kuroiwa et al, NatureGenetics 2004, June 6. The selected cells may then be fused withenucleated nuclear transfer unit cells, e.g. oocytes or embryonic stemcells, cells which are totipotent and capable of forming a functionalneonate. Fusion is performed in accordance with conventional techniqueswhich are well established. Enucleation of oocytes and nuclear transfercan also be performed by microsurgery using injection pipettes. (See,for example, Wakayama et al., Nature (1998) 394:369.) The resulting eggcells are then cultivated in an appropriate medium, and transferred intosynchronized recipients for generating transgenic animals.Alternatively, the selected genetically modified cells can be injectedinto developing embryos which are subsequently developed into chimericanimals.

Further, according to the present invention, a transgenic animal capableof producing human(ized) immunoglobulins can also be made by introducinginto a recipient cell or cells, one or more of the recombination vectorsdescribed herein above, one of which carries a human Ig gene segment,linked to 5′ and 3′ flanking sequences that are homologous to theflanking sequences of the endogenous Ig gene segment, then selectingcells in which the endogenous Ig gene segment is replaced by the humanIg gene segment by homologous recombination, and deriving an animal fromthe selected genetically modified recipient cell or cells.

Similar to the target insertion of a transgenic vector, cellsappropriate for use as recipient cells in this approach includeembryonic stem cells or already differentiated somatic cells. Arecombination vector carrying a human Ig gene segment can be introducedinto such recipient cells by any feasible means, e.g., transfection.Afterwards, cells in which the human Ig gene segment has replaced thecorresponding endogenous Ig gene segment by homologous recombination,can be selected by standard methods. These genetically modified cellscan serve as nuclei donor cells in a nuclear transfer procedure forcloning a transgenic animal. Alternatively, the selected geneticallymodified embryonic stem cells can be injected into developing embryoswhich can be subsequently developed into chimeric animals.

In a specific embodiment, the transgene constructs of the invention maybe introduced into the transgenic animals during embryonic life bydirectly injecting the transgenes into the embryo or indirectly byinjecting them into the pregnant mother or into the egg-laying hen. As aconsequence, due to the inhibition of apoptosis in exogenous B-cells,transgenic offspring will have increased production of human(ized)antibodies in response to immunization with antigens.

Transgenic animals produced by any of the foregoing methods form anotherembodiment of the present invention. The transgenic animals have atleast one, i.e., one or more, human(ized) Ig loci in the genome, fromwhich a functional repertoire of human(ized) antibodies is produced.

In a specific embodiment, the present invention provides transgenicrabbits expressing one or more human(ized) Ig loci and anapoptosis-inhibitor gene. The transgenic rabbits of the presentinvention are capable of rearranging and gene converting the human(ized)Ig loci, and expressing a functional repertoire of human(ized)antibodies.

In another specific embodiment, the present invention providestransgenic chickens expressing one or more human(ized) Ig loci and aapoptosis-inhibitor gene. The transgenic chickens of the presentinvention are capable of rearranging and gene converting the human(ized)Ig loci, and expressing a functional repertoire of human(ized)antibodies. In another specific embodiment, the present inventionprovides transgenic mice expressing one or more human(ized) V regionsand the rabbit bcl-2 apoptosis-inhibitor gene. The human(ized) V regioncomprises at least two human V gene segments flanked by non-human spacersequences. The transgenic mice are capable of rearranging the human Velements and expressing a functional repertoire of antibodies.

Immunization with antigen leads to the production of human(ized)antibodies against the same antigen in said transgenic animals.

Although preferred embodiments of the present invention are directed totransgenic animals having human(ized) Ig loci, it is to be understoodthat transgenic animals having primatized Ig loci and primatizedpolyclonal antisera are also within the spirit of the present invention.Similar to human(ized) polyclonal antisera compositions, primatizedpolyclonal antisera compositions are likely to have a reducedimmunogenicity in human individuals.

Once a transgenic non-human animal capable of producing diversifiedhuman(ized) immunoglobulin molecules is made (as further set forthbelow), human(ized) immunoglobulins and human(ized) antibodypreparations against an antigen can be readily obtained by immunizingthe animal with the antigen. A variety of antigens can be used toimmunize a transgenic host animal. Such antigens include, microorganism,e.g. viruses and unicellular organisms (such as bacteria and fungi),alive, attenuated or dead, fragments of the microorganisms, or antigenicmolecules isolated from the microorganisms.

Preferred bacterial antigens for use in immunizing an animal includepurified antigens from Staphylococcus aureus such as capsularpolysaccharides type 5 and 8, recombinant versions of virulence factorssuch as alpha-toxin, adhesin binding proteins, collagen bindingproteins, and fibronectin binding proteins. Preferred bacterial antigensalso include an attenuated version of S. aureus, Pseudomonas aeruginosa,enterococcus, enterobacter, and Klebsiella pneumoniae, or culturesupernatant from these bacteria cells. Other bacterial antigens whichcan be used in immunization include purified lipopolysaccharide (LPS),capsular antigens, capsular polysaccharides and/or recombinant versionsof the outer membrane proteins, fibronectin binding proteins, endotoxin,and exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,and Klebsiella pneumoniae.

Preferred antigens for the generation of antibodies against fungiinclude attenuated version of fungi or outer membrane proteins thereof,which fungi include, but are not limited to, Candida albicans, Candidaparapsilosis, Candida tropicalis, and Cryptococcus neoformans.

Preferred antigens for use in immunization in order to generateantibodies against viruses include the envelop proteins and attenuatedversions of viruses which include, but are not limited to respiratorysynctial virus (RSV) (particularly the F-Protein), Hepatitis C virus(HCV), Hepatits B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

Therapeutic antibodies can be generated for the treatment of cancer byimmunizing transgenic animals with isolated tumor cells or tumor celllines; tumor-associated antigens which include, but are not limited to,Her-2-neu antigen (antibodies against which are useful for the treatmentof breast cancer); CD19, CD20, CD22 and CD53 antigens (antibodiesagainst which are useful for the treatment of B-cell lymphomas), (3)prostate specific membrane antigen (PMSA) (antibodies against which areuseful for the treatment of prostate cancer), and 17-1A molecule(antibodies against which are useful for the treatment of colon cancer).

The antigens can be administered to a transgenic host animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

After immunization, serum or milk from the immunized transgenic animalscan be fractionated for the purification of pharmaceutical gradepolyclonal antibodies specific for the antigen. In the case oftransgenic birds, antibodies can also be made by fractionating eggyolks. A concentrated, purified immunoglobulin fraction may be obtainedby chromatography (affinity, ionic exchange, gel filtration, etc.),selective precipitation with salts such as ammonium sulfate, organicsolvents such as ethanol, or polymers such as polyethyleneglycol.

The fractionated human(ized) antibodies may be dissolved or diluted innon-toxic, non-pyrogenic media suitable for intravenous administrationin humans, for instance, sterile buffered saline.

The antibody preparations used for administration are generallycharacterized by having immunoglobulin concentrations from 0.1 to 100mg/ml, more usually from 1 to 10 mg/ml. The antibody preparation maycontain immunoglobulins of various isotypes. Alternatively, the antibodypreparation may contain antibodies of only one isotype, or a number ofselected isotypes.

For making a human(ized) monoclonal antibody, spleen cells are isolatedfrom the immunized transgenic animal whose B-cells expressing theanimal's endogenous immunoglobulin have been depleted. Isolated spleencells are used either in cell fusion with transformed cell lines for theproduction of hybridomas, or cDNAs encoding antibodies are cloned bystandard molecular biology techniques and expressed in transfectedcells. The procedures for making monoclonal antibodies are wellestablished in the art. See, e.g., European Patent Application 0 583 980A1 (“Method For Generating Monoclonal Antibodies From Rabbits”), U.S.Pat. No. 4,977,081 (“Stable Rabbit-Mouse Hybridomas And SecretionProducts Thereof”), WO 97/16537 (“Stable Chicken B-cell Line And Methodof Use Thereof”), and EP 0 491 057 B1 (“Hybridoma Which Produces AvianSpecific Immunoglobulin G”), the disclosures of which are incorporatedherein by reference. In vitro production of monoclonal antibodies fromcloned cDNA molecules has been described by Andris-Widhopf et al.,“Methods for the generation of chicken monoclonal antibody fragments byphage display”, J Immunol Methods 242:159 (2000), and by Burton, D. R.,“Phage display”, Immunotechnology 1:87 (1995), the disclosures of whichare incorporated herein by reference.

In most instances the antibody preparation consists of unmodifiedimmunoglobulins, i.e., human(ized) antibodies prepared from the animalwithout additional modification, e.g., by chemicals or enzymes.Alternatively, the immunoglobulin fraction may be subject to treatmentsuch as enzymatic digestion (e.g. with pepsin, papain, plasmin,glycosidases, nucleases, etc.), heating, etc, and/or furtherfractionated.

Embodiments of the invention are directed to transgenes comprising therabbit bcl-2 apoptosis-inhibitor which is expressed specifically inB-cells using a B-cell specific promoter. Another embodiment is directedto transgenes comprising the Ig locus-self-cleaving peptide-apoptosisinhibitor transgene, where expression of the apoptosis inhibitor gene iscoupled to the expression of the Ig locus. Various apoptosis-inhibitorgenes described above and those known in the art, including the rabbitbcl-2 apoptosis inhibitor, can be used in this embodiment.

Further embodiments of the invention are directed to methods to enhancethe survival of B-cells using the transgene constructs described above.When the rabbit bcl-2 transgenic construct is used, the transgene can beintroduced into a transgenic animal further comprising a transgeneencoding an immunoglobulin locus thereby specifically enhancing thesurvival of B-cells. When the Ig locus-self-cleaving peptide-apoptosisinhibitor transgene is used, exogenous B-cells selectively survive andproductively produce the transgene encoded gene. Selectivity is achievedby coupling exogenous immunoglobulin expression with apoptosis inhibitorexpression. In endogenous B-cells, the apoptosis inhibitor is notexpressed and hence, apoptosis is not inhibited. Such selectiveexpression results in the preferential production of the desiredtransgene expressed immunoglobulin over the endogenously producedimmunoglobulin of the transgenic animal. Any variety ofapoptosis-inhibitors, self-cleaving peptides or immunoglobulin genesdescribed herein or well-known in the art can be used in this transgeneconstruct. In a preferred embodiment, the Ig locus of the transgene is ahuman(ized) immunoglobulin/immunoglobulin chain translocus.

The invention also provides a novel apoptosis-inhibitor, rabbit bcl-2,which is useful for enhancing cell survival.

In one aspect of this invention, the non-human transgenic animal whichexpresses the rabbit bcl-2 apoptosis inhibitor is preferably an animalundergoing short-term lymphopoietic B-cell development discussed above,which includes, but is not limited to, animals like rabbits, chickens,sheep and cows, etc. Since these animals are larger, their antibodyproduction and yields, using the methods described above, are alsogreater. In another aspect of the invention, the non-human transgenicanimal which expresses the Ig locus-self-cleaving peptide-apoptosisinhibitor, is any animal including rodents (e.g. mice, rats), rabbits,birds (e.g. chickens, turkeys, ducks, geese, etc.), cows, pigs, sheep,goats, horses, donkeys and other farm animals. In a further aspect, thetransgenic animals used in the methods of the invention can either begene converting animals or animals that can undergo antibodydiversification by gene rearrangement that stops early in life. In apreferred embodiment, the non-human transgenic animal is the rabbit.

Thus, the transgenic constructs, the vectors comprising the transgeneconstructs and the transgenic animals generated using the methodsdescribed above are all embodiments of the invention.

The invention is further illustrated, but by no means limited, by thefollowing examples.

EXAMPLE 1

Construction of a Apoptosis-Inhibitor Expression Vector with Human bcl-2

Screening of rabbit genomic BAC libraries resulted in the identificationof two BACs (179L1 and 19602; Gene Bank Accession Nos: AY495827, andAY495828, respectively) containing rabbit light chain K1 gene segments.

For the construction of a B-cell specific apoptosis-inhibitor expressionvector, BAC AY495827 was modified by homologous recombination in E. coli(ET cloning: E. Chiang Lee et al., Genomics 73, 56-65 (2001); Daiguan Yuet al., PNAS 97, 5978-5983 (2000); Muyrers et al., Nucleic AcidsResearch 27, 1555-1557 (1999); Zhang et al., Nature Biotechnology 18,1314-1317(2000)) and nucleotides 1-107795 and 142832-205141 weredeleted. A synthetic human bcl-2 gene, under control of the kappa 1promoter from AY495828 (pos. 114284-114570) further connected to therabbit beta globin polyA sequence, was synthesized. Downstream, agentamycin selection cassette flanked by FRT-sites was introduced byoverlap extension PCR. The bcl-2-gentamycin cassette was amplified withprimers having 50 bp homologies to the modified AY495827 BAC (SEQ ID NO:1). The sequence from nucleotide 134571-136019 on BAC AY495827 wasexchanged against the bcl-2-gentamycin cassette (SEQ ID NO: 1) by ETcloning. Positive clones were selected with gentamycin, analyzed byrestriction enzyme digests and confirmed by sequencing. Subsequently,the gentamycin selection cassette was removed by FLP-recombination. Theresulting construct was used to generate transgenic animals.

EXAMPLE 2

Construction of a Apoptosis-Inhibitor Expression Vector with Mouse bcl-2

Screening of a rabbit genomic BAC libraries resulted in theidentification of two BACs (179L1 and 196O2; Gene Bank Accession Nos:AY495827, and AY495828, respectively) containing rabbit light chain K1gene segments.

For the construction of a B-cell specific apoptosis-inhibitor expressionvector, BAC AY495827 was modified by homologous recombination in E. coli(ET cloning: (E. Chiang Lee et al., Genomics 73, 56-65 (2001); DaiguanYu et al., PNAS 97, 5978-5983 (2000); Muyrers et al., Nucleic AcidsResearch 27, 1555-1557 (1999); Zhang et al., Nature Biotechnology 18,1314-1317(2000) and nucleotides 1-107795 and 142832-205141 were deleted.A synthetic mouse bcl-2 gene under the control of the kappa 1 promoterfrom AY495828 (pos. 114284-114570), further connected to the rabbit betaglobin polyA sequence, was synthesized. Downstream, a gentamycinselection cassette flanked by FRT-sites was introduced by overlapextension PCR. The bcl-2-gentamycin cassette was amplified with primershaving 50 bp homologies to the modified AY495827 BAC (SEQ ID NO:1). Thesequence from nucleotide 134571-136019 on BAC AY495827 was exchangedagainst the bcl-2-gentamycin cassette by ET cloning. Positive cloneswere selected with gentamycin and analyzed by restriction enzyme digestsand confirmed by sequencing. Subsequently, the gentamycin selectioncassette was removed by FLP-recombination. The resulting construct wasused to generate transgenic animals.

EXAMPLE 3

Construction of a Human(ized) Heavy Chain Locus Encoding a FusionProtein Consisting of the Membrane Forms of IgM and IgG. a 2ASelf-Cleaving Peptide, and Apoptosis-Inhibitor

BAC and fosmid clones containing rabbit immunoglobulin heavy chain locussequences were isolated from genomic DNA libraries using probes specificfor the constant, variable, and joining gene segments or the 3′ enhancerregion. Isolated BACs and fosmid Fos15B were sequenced (Genebank acc.No. AY386695, AY386696, AY386697, AY386698). The J and Cμ regions ofAY386695 and the C□ region of AY386696 were exchanged with correspondinghuman counterparts by homologous recombination in E. coliby ET cloning(E. Chiang Lee et al., Genomics 73, 56-65 (2001); Daiguan Yu et al.,PNAS 97, 5978-5983 (2000); Muyrers et al., Nucleic Acids Research 27,1555-1557 (1999); Zhang et al., Nature Biotechnology 18,1314-1317(2000)).

The four BACs were recombined by in vitro ligation and Cre-mediatedrecombination to reconstitute a rabbit Ig locus with human J, Cμ and C□coding sequences.

To link the expression of bcl-2 to the expression of IgM and IgG, thecoding sequence of bcl-2 was fused with the coding sequence of M2membrane exons of IgM and IgG with a sequence coding for a F2Aself-cleaving peptide.

For the insertion of a sequence encoding an IgG-M2-F2A-bcl-2 fusionprotein the following construct was generated. Sequences for homologousrecombination were based on the sequence of BAC AY 386696. A DNAfragment (from 5′ to 3′) containing a KpnI site, a sequence identical to50 nucleotides of Cγ M2, a sequence encoding F2A, a sequence encoding ahuman bcl-2, an FRT5 site, and an EcoRI site was synthesized. TherpsL.Neo counter selection cassette was amplified using plasmid pSC101rpsL-neo (Genebridges) as template. The upstream primer contained aEcoRI and an FRT5 site, the downstream primer contained a sequenceidentical to 50 nucleotides downstream of Cγ M2 and a XhoI site. Thesynthetic fragment and the PCR amplification product were ligated intothe pcDNA3.1(+) vector, opened with KpnI and XhoI. The ligated cassette(SEQ ID NO: 2) was released with XhoI and KpnI and used for homologousrecombination in E coli. Following transformation of the cassette intothe E. coli strain DH10B containing BAC AY 386696 and the plasmidpSC101-BAD-gba-tetra, expression of recombinases Redα/β was induced.Subsequently, kanamycin resistant clones were selected and were analysedby restriction enzyme digestion and partial sequence analysis. Lastly,the RpsLNeo-resistance gene was deleted from the BAC by Flp-mediatedrecombination.

The resulting BAC clone was further modified through insertion of asequence encoding an IgM-M2-F2A-bcl-2 fusion protein. Sequences forhomologous recombination are based on the sequence of BAC AY 386696. TherpsL.Neo gene was amplified using plasmid pSC101 rpsL-neo (Genebridges)as the template. Primers contain sequences identical to IgG-M2 andflanking sequences, FRT- and FRT2-sites (SEQ ID NO: 3). Theamplification product was inserted into BAC AY 386696 by ET cloning.Subsequently, the selection cassette was replaced with a DNA fragmentencoding an IgM-M2-F2A-bcl-2 fusing protein (SEQ ID NO: 4). This DNAfragment was synthesized containing from 5′ to 3′-an EcoRI site, an FRTsite, a sequence encoding IgM-M2, a sequence encoding F2A and bcl-2, aFRT2 and an EcoRI site (SEQ ID NO: 4). The synthesized fragment wasreleased with EcoRI and was used for the exchange of the rpsL.Neo genewith IgM-M2-F2A-bcl-2 by Flp-mediated recombination between FRT/FRT andFRT2/FRT2 sites. Positive clones are identified by restriction analysisand further analysed by partial sequencing.

The resulting BAC was combined with BACs containing different V-regions.BACs can be combined by ligation or recombination. The resultingconstructs were used for the generation of transgenic animals.

EXAMPLE 4

Generation of Transgenic Mice and Rabbits Expressing Humanized HeavyChain Immunoglobulins

Transgenic rabbits and mice containing humanized heavy and light chainimmunoglobulin loci and a apoptosis-inhibitor gene are generated byinjection of DNA into the pronuclei of fertilized oocytes and subsequenttransfer of embryos into foster mothers. Transgenic founder animals areidentified by PCR. Expression of human(ized) immunoglobulin M and G ismeasured by ELISA. Expression of humanized IgG was 10-20 mg/ml.

EXAMPLE 5

Generation of Transgenic Chicken Expressing Humanized Heavy ChainImmunoglobulins

Transgenic chicken were generated by testis mediated gene transfer. DNAconstructs (50ug) are mixed with 250ul lipofection reagent (superfect)in 500ul 0.9% NaCl and injected in the testis of roosters. Three to fourweeks later roosters with transgenic sperm are identified by PCRanalysis and mated with hens. Transgenic offspring were identified byPCR. Expression of humanized IgG is 10-20 mg/ml.

All references cited throughout the disclosure along with referencescited therein are hereby expressly incorporated by reference.

While the invention is illustrated by reference to certain embodiments,it is not so limited. One skilled in the art will understand thatvarious modifications are readily available and can be performed withoutsubstantial change in the way the invention works. All suchmodifications are specifically intended to be within the scope of theinvention claimed herein.

1. A polypeptide comprising the rabbit bcl-2 polypeptide of SEQ ID NO:5.
 2. The polypeptide of claim 1 wherein said bcl-2 polypeptide of SEQID NO: 5 is fused to a heterologous amino acid sequence.
 3. Thepolypeptide of claim 2 wherein said heterologous amino acid sequence isan epitope sequence.
 4. The polypeptide of claim 2 wherein saidheterologous amino acid sequence is an immunoglobulin sequence.
 5. Thepolypeptide of claim 4 wherein said immunoglobulin sequence is an Fcregion of an immunoglobulin.
 6. An isolated nucleic acid moleculecomprising a sequence encoding the rabbit bcl-2 polypeptide of SEQ IDNO:
 5. 7. A vector, expression cassette or transgenic expressionconstruct comprising the nucleic acid molecule of claim
 6. 8. Thetransgenic expression construct of claim 7 wherein expression of saidrabbit bcl-2 apoptosis inhibitor is driven by a B-cell specificpromoter/enhancer.
 9. The transgenic expression construct of claim 8wherein said B-cell specific promoter/enhancer is selected from thegroup consisting of a promoter/enhancer of CD19, CD20, CD21, CD22, CD23,CD24, CD40, CD72, Blimp-1, CD79b, mb-1, tyrosine kinase blk, VpreB,immunoglobulin heavy chain, immunoglobulin kappa light chain,immunoglobulin lambda light chain and immunoglobulin J-chain genes ormodifications thereof.
 10. An isolated host cell transformed with thevector, expression cassette or transgenic expression construct of claim7.
 11. A method for enhancing the expression of an immunoglobulin orimmunoglobulin chain in a transgenic animal undergoing short-termlymphopoiesis, comprising introducing into said transgenic animalundergoing short-term lymphopoiesis at least one transgene constructcomprising an apoptosis-inhibitor transgene driven by a B-cell specificpromoter/enhancer to produce said transgenic animal, whereby apoptosisof the B-cells carrying said transgene construct is inhibited andproduction of the immunoglobulin or immunoglobulin chain is enhanced.12. The method of claim 11 wherein said transgenic animal is selectedfrom the group consisting of rabbits, birds, chickens, sheep, goats,cows, swine, horses and donkeys.
 13. The method of claim 11 wherein saidtransgenic animal is a rabbit.
 14. The method of claim 11 wherein saidB-cell specific promoter/enhancer is selected from the group consistingof CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, Blimp-1, CD79b, mb-1,tyrosine kinase blk, VpreB, immunoglobulin kappa light chain,immunoglobulin lambda light chain and immunoglobulin J-chain promotersor modifications thereof.
 15. The method of claim 11 wherein saidapoptosis-inhibitor is selected from the group consisting of bcl-2,caspase-9-DN mutants, baculovirus p35, caspase-9S, crmA, z-VAD-fink,z-DEVD-fmk, B-D-fmk, z-YVAD-fmk, Bcl-x_(L), Mcl-1, XIAP, TIAP, KIAP,NAIP, cIAP1, cIAP2, API1, API2, API3, API4, HIAP1, HIAP2, MIHA, MIHB,MIHC, ILP, ILP-2, TLAP, survivin, livin, apollon, BRUCE, MLIAP, SODD andFLIP and variants thereof.
 16. The method of claim II further comprisingintroducing into said transgenic animal undergoing short-termlymphopoiesis at least one more transgene encoding for an exogenousimmunoglobulin or immunoglobulin chain transgene locus.
 17. The methodof claim 16 wherein said exogenous immunoglobulin or immunoglobulinchain is a human(ized) immunoglobulin heavy and/or light chain sequence.18. The method of claim 17 wherein the exogenous immunoglobulin orimmunoglobulin chain transgene locus and the apoptosis-inhibitortransgene are both present on the same transgenic expression vector. 19.The method of claim 17 wherein the exogenous immunoglobulin orimmunoglobulin chain transgene locus and the apoptosis-inhibitortransgene are present on different transgenic expression vectors. 20.The method of claim 19 wherein each of said transgenic expressionvectors are introduced into the transgenic animal at the same time. 21.The method of claim 19 wherein each of said transgenic expressionvectors are introduced into the transgenic animal sequentially.
 22. Anon-human transgenic animal undergoing short-term lymphopoiesiscomprising at least one transgene construct comprising an apoptosisinhibitor transgene driven by a B-cell specific promoter/enhancer.
 23. Anon-human transgenic animal undergoing short-term lymphopoiesiscomprising at least one transgene construct comprising an apoptosisinhibitor transgene driven by a B-cell specific promoter/enhancer and atleast one exogenous immunoglobulin or immunoglobulin chain transgenelocus.
 24. The non-human transgenic animal of claim 23 wherein saidexogenous immunoglobulin or immunoglobulin chain is a human(ized)immunoglobulin heavy and/or light chain sequence.
 25. The non-humantransgenic animal of claim 22 or 23 selected from the group consistingof rabbits, birds, chickens, sheep, goats, cows, swine, horses anddonkeys.
 26. A transgenic expression construct comprising a transgeneencoding a fusion-protein comprising polypeptide sequences in thefollowing order: a) an immunoglobulin or immunoglobulin chain; b) aself-cleaving peptide; c) an apoptosis inhibitor; and optionally, d) aprotease cleavage site between a) and b).
 27. The transgenic expressionconstruct of claim 26 wherein said protease cleavage site is selectedfrom the group consisting of sites of aspartic proteases, cysteineproteases, metalloproteases, serine proteases and threonine proteases.28. The transgenic expression construct of claim 26 wherein saidprotease cleavage site is the furin cleavage site.
 29. The transgenicexpression construct of claim 26 wherein said immunoglobulin fragment isa fragment of a human(ized) immunoglobulin heavy and/or light chain. 30.The transgenic expression construct of claim 26 wherein saidself-cleaving peptide is derived from viral 2A/2B or 2A-like/2Bpeptides.
 31. The transgenic expression construct of claim 30 whereinsaid virus is selected from the group consisting of the picornaviridaevirus family, the equine rhinitis A (ERAV) virus family, thepicomavirus-like insect virus family and from the type C rotavirusfamily.
 32. The transgenic expression construct of claim 31 wherein saidvirus is selected from the group consisting of the foot and mouthdisease virus (FMDV), the equine rhinitis A (ERAV) virus, and the Thoseaasigna virus (TaV).
 33. The transgenic expression construct of claim 26wherein said apoptosis inhibitor is selected from the group consistingof bcl-2, caspase-9-DN mutants, baculovirus p35, caspase-9S, crmA,z-VAD-fmk, z-DEVD-fmk, B-D-fmk, z-YVAD-fmk, Bcl-x_(L), Mcl-1, XIAP,TIAP, KIAP, NAIP, cIAP1, cIAP2, API1, API2, API3, API4, HIAP1, HIAP2,MIHA, MIHB, MIHC, ILP, ILP-2, TLAP, survivin, livin, apollon, BRUCE,MLIAP, SODD and FLIP and variants thereof.
 34. The transgenic expressionconstruct of claim 26 wherein said apoptosis inhibitor is mammalianbcl-2.
 35. The transgenic expression construct of claim 34 wherein saidmammalian bcl-2 is selected from the group consisting of human bcl-2,mouse bcl-2 and rabbit bcl-2.
 36. An isolated host cell transformed withthe transgenic expression construct of claim
 26. 37. A method forselectively enhancing the expression of an exogenous immunoglobulin orimmunoglobulin chain within an exogenous B-cell of a non-humantransgenic animal, comprising introducing into said animal a transgeneconstruct encoding a fusion-protein comprising polypeptide sequences inthe following order: a) an immunoglobulin or immunoglobulin chain; b) aself-cleaving peptide; c) an apoptosis inhibitor, and, optionally; d) aprotease cleavage site between a) and b), whereby survival of theexogenous B cell and exogenous immunoglobulin production are enhanced.38. The method of claim 37 wherein said exogenous immunoglobulin orimmunoglobulin chain is a human(ized) immunoglobulin heavy and/or lightchain.
 39. The method of claim 37 wherein said protease cleavage site isselected from the group consisting of sites of aspartic proteases,cysteine proteases, metalloproteases, serine proteases and threonineproteases.
 40. The method of claim 37 wherein said protease cleavagesite is the furin cleavage site.
 41. The method of claim 37 wherein saidgene of a self-cleaving peptide is obtained from viral 2A/2B or2A-like/2B.
 42. The method of claim 41 wherein said virus is selectedfrom the group consisting of the picornaviridae virus family, the equinerhinitis A (ERAV) virus family, the picomavirus-like insect virus familyand from the type C rotavirus family.
 43. The method of claim 41 whereinsaid virus is selected from the group consisting of the foot and mouthdisease virus (FMDV), the equine rhinitis A (ERAV) virus, and the Thoseaasigna virus (TaV).
 44. The method of claim 37 wherein saidapoptosis-inhibitor is selected from the group consisting of bcl-2,caspase-9-DN mutants, baculovirus p35, caspase-9S, crmA, z-VAD-fmk,z-DEVD-fmk, B-D-fmk, z-YVAD-fink, Bcl-x_(L), Mcl-1, XIAP, TIAP, KIAP,NAIP, cIAP1, cIAP2, API1, API2, API3, API4, HIAP1, HIAP2, MIHA, MIHB,MIHC, ILP, ILP-2, TLAP, survivin, livin, apollon, BRUCE, MLIAP, SODD andFLIP and variants thereof.
 45. The method of claim 37 wherein saidnon-human transgenic animal is selected from the group consisting ofrodents, primates, rabbits, birds, cows, pigs, sheep, goats, horses anddonkeys.
 46. A non-human transgenic animal comprising at least onetransgene construct encoding a fusion-protein comprising polypeptidesequences in the following order: a) an immunoglobulin or immunoglobulinchain; b) a self-cleaving peptide; c) an apoptosis inhibitor, and,optionally; d) a protease cleavage site between a) and b), whereby saidfusion-protein is expressed
 47. The non-human transgenic animal of claim46 wherein said animal is selected from the group consisting of rodents,primates, rabbits, birds, cows, pigs, sheep, goats, horses and donkeys.